Oligosaccharides as antimicrobials and prebiotics Carbohydrates on human intestinal cell surface are important recognition sites for pathogenic bacterial binding that initiates infection. They are also the major components of human milk oligosaccharides. These carbohydrates are often terminated with a galactose, a fucose, or a sialic acid residue. Since human milk oligosaccharides cannot be metabolized by infants, they are believed to function as prebiotics that stimulate the growth and proliferation of beneficial bacteria whose colonization in the human intestine competes with pathogenic bacteria for binding. Presumably they also mimic the carbohydrate structures on human intestinal surface, and thus act as antimicrobial decoys that deflect pathogenic bacteria away from human intestinal surface. Although evidence is accumulating to support these hypotheses, it is unclear whether beneficial and pathogenic bacteria compete for the same types of carbohydrates for binding on human intestinal surface. It is also not clear whether a single human milk oligosaccharide or a mixture of two or more oligosaccharides is required for these presumed beneficial effects of human milk. We hypothesize that the carbohydrate structures that exist both in human milk and on the surface of human intestinal cells are the most suitable antimicrobial and prebiotic candidates. We propose to develop effective synthetic methods for efficient production of human milk and human cell surface oligosaccharides with well-defined structures and in amounts large enough for multiple types of lab research. The synthesized oligosaccharides will be screened for bacterial binding and inhibition studies [as individual compounds and as mixtures]. They will be further used individually or in subsets in bacteria growth studies to identify the key antimicrobials and prebiotics. [The potential synergistic effect of mixtures of oligosaccharides will also be analyzed.] The specific aims of this proposed project are as follows: (1) to develop methods for gram-scale synthesis of galactosides as well as sialylated and/or fucosylated oligosaccharides; (2) to develop methods and assays for bacterial binding and inhibition studies for pathogens, commensals, and probiotics; and (3) to perform bacterial growth studies using synthesized oligosaccharides and perform functional studies to identify potential mechanisms for antimicrobial or prebiotic effects. We have a multi-disciplinary research team comprised of a chemist (Chen), a physicist (Zhu), a nutritionist (German), and microbiologists (Mills, Weimer) with complementary expertise to achieve these specific aims. We will produce large quantity of pure, structurally complex carbohydrates, and from these pure compounds [as well as their mixtures of known composition], we will identify key components in human milk oligosaccharides that are responsible for the probiotic growth-promoting property. We will address the question as to whether probiotics and pathogenic bacteria compete for the same types of carbohydrates on human intestinal surface. Potential carbohydrate antimicrobials and prebiotics will be identified. PUBLIC HEALTH RELEVANCE: Oligosaccharides as antimicrobials and prebiotics Human milk oligosaccharides are believed to promote the growth of beneficial bacteria and act as inhibitors or decoys against pathogenic bacteria binding to the human intestinal surface. The ubiquitous occurrence and the presumed biological role of human milk oligosaccharides and the recognized difficulties in obtaining large amounts of these compounds in pure and diverse forms motivate our proposed synthesis of these compounds in large quantity for multiple research purposes. With the availability of these compounds, we will elucidate the mechanisms behind their presumed beneficial effects by performing a comprehensive set of binding, growth, and inhibition studies, and in turn identify potent antimicrobial and prebiotic carbohydrates.